The mechanisms of transcription initiation and its regulation will be studied in bacteriophage lambda. These studies will concentrate on the right operator-promoter complex of lambda. This DNA region contains two promoters and multiple repressor binding sites which direct transcription both to the right and to the left from this region. Products of two lambda genes, cI and cro regulate the initiation of these transcripts. This project will study the structure of this region by isolating mutations affecting this region and analyzing them with nucleotide sequencing, physiological, and genetic techniques. Mutations in the operator-promoter complex will be isolated and mapped with a set of deletion mutations, also to be isolated. The nucleotide changes in the mutations will be determined by the DNA sequencing techniques developed by Sanger and his colleagues. The physiological alterations of these mutants will be compared with their nucleotide changes to determine the role of the individual sequences within the complex site. The results will be used to develop models for the regulation of bi-directional transcription and protein-nucleic acid interaction. The genetic and DNA sequence analysis techniques developed for our studies of the site of transcription initiation will also be used to study special sites involved in generalized recombination. These sites are the Chi recombinational hotspots of lambda studied by F. Stahl and his collaborators, with whom we are collaborating. We will determine the nucleotide sequence of several such sites to determine if there is a common sequence which is responsible for the recombination hotspot phenotype. We also plan to search for a protein involved in recombination which acts at these sites, thereby determining how Chi sites stimulate recombination.